The intestinal fatty acid binding protein is one of a family of proteins that are composed of two beta-sheets surrounding a large interior cavity into which the ligand binds. Glycine residues occur in many of the turns between adjacent antiparallel beta-strands. In previous work, the effect of replacing these glycine residues with valine has been examined with stopped flow instrumentation using intrinsic tryptophan fluorescence spectroscopy [Kim and Frieden (1998) Protein Sci. 7, 1821-1828]. To resolve the burst phase missing in the stopped flow measurements, these valine mutants have been reexamined with sub-millisecond continuous flow instrumentation. Some of the glycine residues have also been replaced with proline, and the folding reactions of these proline mutants have been compared with those of their valine counterparts. In all cases, the stability of the protein is decreased, but some turns appear to be more critical for final structure stabilization than others. Surprisingly, the rate constants observed for all the mutants measured by sub-millisecond continuous flow methods are quite similar (1400-3000 s(-1)), and in all the mutants, there is a shift in the fluorescence emission maximum from that of the unfolded protein to lower wavelengths, suggesting some collapse of the unfolded state within 200 micros. In contrast to the rate constants observed for the initial folding events measured by the sub-millisecond continuous flow method, the rate constants for the slower phase observed in the stopped flow instrument vary widely for the different mutants. The latter step appears to be related to side chain stabilization rather than secondary structure formation. It is also shown that the ligand binds tightly only to the native protein and not to any intermediate forms.

The folding reaction of acid-unfolded cytochrome c in the presence of various amounts of KCl was investigated with Trp fluorescence and resonance Raman spectroscopies. It was found that the too-early-too-much polypeptide chain collapse induced by KCl yields some stable folding intermediates, which need to overcome a higher energy barrier to fold into their native conformation. We propose that the charge distribution on the polypeptide chain is part of the folding codon encoded in the linear amino acid sequence. The charge screening effect introduced by KCl alters the shape of the energy landscape by raising the slope of the upper rim and introduces a rugged energy surface toward the bottom of the folding funnel.

Nanosized aluminum-doped zinc oxide Zn1−xAlxO (AZO) powders (AZO-NPs) with x = 0.01, 0.03, 0.06, 0.09 and 0.11 were synthesized by chemical precipitation method. The thermogravimetric analysis (TGA) indicated that the precursors were converted to oxides from hydroxides near 250 °C, which were then heated to 500 °C for subsequent thermal processes to obtain preliminary powders. The obtained preliminary powders were then calcined at 500 °C for three hours. The structure and morphology of the products were measured and characterized by angle-dispersive X-ray diffraction (ADXRD) and scanning electron microscopy (SEM). ADXRD results showed that AZO-NPs with Al content less than 11% exhibited würtzite zinc oxide structure and there was no other impurity phase in the AZO-NPs, suggesting substitutional doping of Al on Zn sites. The Zn0.97Al0.03O powders (A3ZO-NPs) with grain size of about 21.4 nm were used for high-pressure measurements. The in situ ADXRD measurements revealed that, for loading run, the pressure-induced würtzite (B4)-to-rocksalt (B1) structural phase transition began at 9.0(1) GPa. Compared to the predicted phase-transition pressure of ~12.7 GPa for pristine ZnO nanocrystals of similar grain size (~21.4 nm), the transition pressure for the present A3ZO-NPs exhibited a reduction of ~3.7 GPa. The significant reduction in phase-transition pressure is attributed to the effects of highly selective site occupation, namely Zn2+ and Al3+, were mainly found in tetrahedral and octahedral sites, respectively.